Polypeptide for the treatment of cancer and a method for preparation thereof

ABSTRACT

This invention characterizes the specific peptide fragment derived from specially prepared zinc charged fetuin and a method of preparation thereof, wherein the fragment was found to contain an apoptosis-inducing activity. Specifically, the amino acid sequence of this peptide is H-T-F-S-G-V-A-S-V-E and correlates to amino acid no. 300-309 of fetuin, referred to herein as Fetuin Peptide Fragment (FPF 300-09). FPF 300-09 strongly induced apoptosis in LNCaP (prostate cancer) and HT-29 (colon cancer) cells without affecting CCD 18 Co (normal colon) cells. The in vitro tissue culture study demonstrated that the FPF 300-09 is more potent than the parent molecule (full-length zinc charged fetuin) in inducing apoptosis. FPF 300-09 has a LD 50  of 0.3-0.4 μM while the LD 50  for zinc-charged fetuin is 3-10 μM.

CLAIM OF PRIORITY

[0001] This application is a continuation-in-part to U.S. Ser. No.09/902,208 (filed 09 Jul. 2001), which is a continuation-in-part to U.S.Ser. No. 09/414,136 (filed 07 Oct. 1999), now U.S. Pat. No. 6,258,779(issued 10 Jul. 2001), which is a continuation-in-part to U.S. Ser. No.09/149,878 (filed 08 Sep. 1998), now U.S. Pat. No. 5,994,298 (issued 30Nov. 1999), which is a continuation-in-part to U.S. Ser. No. 08/993,432(filed 18 Dec. 1997).

BACKGROUND OF THE INVENTION

[0002] Human beings have had a long battle against cancer. Because thedisease is so widespread, manifests itself in so many different ways andis so relentless, the potential market for effective cancer therapies isenormous. It is estimated that 10 million people in the U.S. either haveor have had cancer. The National Cancer Institute (NCI) projects that in1995, some 1.2 million new cases of cancer will be diagnosed in theUnited States, and that 538,000 people will die of the disease. Canceris currently treated, with a low degree of success, with combinations ofsurgery, chemotherapy and radiation. The reason of the low degrees ofsuccess in cancer chemotherapy is as the following: currentchemotherapeutic approaches target rapidly dividing tumor cells. Thisapproach is ineffective when the cancer is dormant or growing slowly.Such treatments also affect other, noncancerous cells that dividerapidly, causing harmful side effects.

[0003] Only in the last several years has a new approach emerged in thebattle against cancer. This approach is based on the newly discoveredbiological phenomenon called “Apoptosis”. Apoptosis is also called“programmed cell death” or “cell suicide”. (Krammer, et al., “Apoptosisin the APO-1 System”, Apoptosis: The molecular Basis of Cell Death, pp.87-99 Cold Spring Harbor Laboratory Press, 1991). In contrast to thecell death caused by cell injury, apoptosis is an active process ofgene-directed, cellular self-destruction and that it serves abiologically meaningful function. (Kerr, J. F. R and J. Searle J.Pathol. 107:41, 1971). One of the examples of the biologicallymeaningful functions of apoptosis is the morphogenesis of embryo.(Michaelson, J. Biol. Rev. 62:115, 1987). Just like the sculpturing of asculpture, which needs the addition as well as removal of clay, theorgan formation (Morphogenesis) of an embryo relies on cell growth(addition of clay) as well as cell death (removal of clay). As a matterof fact, apoptosis plays a key role in the human body from the earlystages of embryonic development through to the inevitable declineassociated with old age. (Wyllie, A. H. Int. Rev. Cytol. 68:251, 1980).The normal function of the immune, gastrointestinal and hematopoieticsystem relies on the normal function of apoptosis. When the normalfunction of apoptosis goes awry, the cause or the result can be one of anumber of diseases, including: cancer, viral infections, auto-immunedisease/allergies, neurodegeneration or cardiovascular diseases. Becauseof the versatility of apoptosis involved in human diseases, apoptosis isbecoming a prominent buzzword in the pharmaceutical research field. Hugeamounts of time and money are being spent in an attempt to understandhow it works, how it can be encouraged or inhibited and what this meansfor practical medicine. A handful of companies have been formed with theprime direction of turning work in this nascent field into marketablepharmaceutical products. The emergence of a core of innovative youngcompanies combined with the tentative steps being taken by establishedindustrial players are certain to make apoptosis research one of thefastest-growing and most promising areas of medical study of the 1990's.

[0004] The idea that cancer may be caused by insufficient apoptosismerged only recently (Cope, F. O. and Wille, J. J., “Apoptosis”: TheMolecular Basis of Cell Death, Cold Spring Harbor Laboratory Press, p.61, 1991). This idea however, opens a door for a new concept in cancertherapy—Cancer cells may be killed by encouraging apoptosis. Apoptosismodulation, based on the processes present in normal development, is apotential mechanism for controlling the growth of tumor cells. Restoringapoptosis in tumor cells is an attractive approach because, at least intheory, it would teach the cells to commit suicide. Nevertheless, sincethe objective of cancer treatment is to kill cancer cells withoutkilling the host, although apoptosis may open a new door for cancertherapy by inducing apoptosis in tumor cells, the success of thistreatment is still dependent on the availability of drugs that canselectively induce apoptosis in tumor cells without affecting normalcells. In this patent application, we described the methods for theisolation of proteins that specifically induce apoptosis in cancer cellswithout effect in normal cells. These proteins may present a new classof anticancer drugs that induce apoptosis in cancer cells, which mayoffer a breakthrough in cancer therapy.

SUMMARY OF THE INVENTION

[0005] The purpose of this invention is to characterize the specificpeptide fragment derived from specially prepared zinc charged fetuinwherein the fragment was found to contain an apoptosis-inducingactivity. Specifically, the amino acid sequence of this peptide isH-T-F-S-G-V-A-S-V-E (His Thr Phe Ser Gly Val Ala Ser Val Glu, SEQ IDNO:1) and correlates to amino acid no. 300-309 of fetuin, referred toherein as Fetuin Peptide Fragment (FPF. 300-09). FPF 300-09 stronglyinduced apoptosis in LNCaP (prostate cancer) and HT-29 (colon cancer)cells without affecting CCD 18 Co (normal colon) cells. The in vitrotissue culture study demonstrated that the FPF 300-09 is more potentthan the parent molecule in inducing apoptosis. FPF 300-09 has a LD₅₀ of0.3-0.4 μM, while the LD₅₀ for zinc-charged fetuin is 3-10 μM.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 shows a table of test data of using fetal fetuin on micebearing leukemia.

[0007]FIG. 2 shows a table of the comparison of original fetuin withzinc in comparison with the supercharged zinc fetuin to reach LD₅₀.

[0008]FIG. 3 shows another table of the comparison of original fetuinwith zinc in comparison with the supercharged zinc fetuin to reach LD₅₀.

[0009]FIG. 4 shows a slide of LNCaP (prostate cancer) cells withouttreatment of filtrate containing FPF 300-09.

[0010]FIG. 5 shows a slide of LNCaP cells incubated with filtratecontaining FPF 300-09 for six (6) hours.

[0011]FIG. 6 shows an additional slide of LNCaP (prostate cancer) cellswithout treatment of filtrate containing FPF 300-09.

[0012]FIG. 7 shows a slide of LNCaP cells incubated with filtratecontaining FPF 300-09 and which expressed membrane bubbling, which is anindicator of cells undergoing apoptosis.

[0013]FIG. 8 shows a table illustrating, among other things, the effectof incubating the filtrate containing FPF 300-09 treated with proteinaseK.

[0014]FIG. 9 shows a slide of LNCaP cells without filtrate containingFPF 300-09.

[0015]FIG. 10 shows a slide of LNCaP cells, which were incubated withfiltrate containing FPF 300-09 for three (3) hours and expressedmembrane “bubbling,” which is an indicator of cells undergoingapoptosis.

[0016]FIG. 11 is a graph of FPF 300-09 (nM) versus the percent apoptosisin HT-29, CCD-18 Co, and LNCaP cells.

[0017]FIG. 12 is a graph of the time of incubation of FPF 300-09filtrate versus percent apoptosis in HT-29 and CCD-18 Co cells.

[0018]FIG. 13 is a table of the LD₅₀ values for fetuin and variousfetuin fragments, including FPF 300-09.

DETAILED DESCRIPTION OF THE INVENTION

[0019] This patent application describes the isolation of five proteins:Apogen P-1a, Apogen 1b, Apogen 1c, Apogen P-2 and Apogen L.

(A) Isolation of Apogen P-1

[0020] (1) Source of Apogen P-1

[0021] Apogen P-1 was isolated from the conditioned medium of a cellline called XC, which was derived from rat tumor (ATCC CCL 165). XCcells were first grown in Dulbecco's Modification of Eagle's Medium(DMEM) containing 10% Fetal bovine serum (FBS) for 3 days. XC cells werethen washed with PBS (3×100 ml) to remove serum and then grown in DMEMcontaining no FBS for 4 days. From this serum free conditioned medium,we detected an activity inducing apoptosis in a prostate cancer cellline called LNCAP. On the other hand, normal human lung fibroblast cellline (CCD 39 Lu) and breast cancer cells (MCF-7) is not affected by thisactivity.

[0022] (2) Activity of Apogen P-1

[0023] (a) Apoptosis Inducing Activity

[0024] The activity of the crude conditioned medium of XC cells wastested on the following cell lines: JEG-3 (Choriocarcinoma), G401(Wilm's tumor) LNCAP (Prostate cancer), T84 (colon cancer), HL-60(leukemia), breast cancer cells (MCF-7), and CCD 39 Lu (normal lungfibroblast). When 10 folds concentrated conditioned medium was incubatedfor 18 hours with the above cell lines in the presence of 5% serum, theconditioned medium induced apoptosis in JEG-3 cells (35%), G 401 cell(27%), LNCaP(100%) and without activity in CCD 39 Lu (0%), T84(0%),MCF-7(0%) and HL-60(0%).

[0025] Apoptosis is a distinct type of cell death that differsfundamentally from degenerative death or necrosis in its nature andbiological significance. A cell undergoing apoptosis is distinct from acell undergoing necrosis both morphologically and biochemically.Morphologically, the earliest definitive changes in apoptosis that havebeen detected with the electron microscope are compaction of the nuclearchromatin into sharply circumscribed, uniformly dense masses about thenuclear envelope and condensation of the cytoplasms. Phase-contrastmicroscope of cells under apoptosis shows the condensation and thefragmentation of DNA and the budding of cell to form apoptotic body.

[0026] To morphologically demonstrate that the XC conditioned mediumcontains activity inducing apoptosis, LNCAP cells were incubated withcontrol medium or the conditioned medium treated as described as abovefor 15 hr and then stained with Hoechst dye for 2 hours. The nuclei ofthe LNCAP cells that have been incubated with the control medium arenormal and healthy. However, the nuclei of the LNCAP cells that havebeen incubated with the conditioned medium (X20, exchanged to RPMI) showthe characteristics of apoptosis. First, the conditioned medium causesthe condensation of the nucleus, demonstrated by the more intensefluorescent light compared with the control nucleus. Secondly, thenucleus condensation is accompanied by the fragmentation of DNA,demonstrated by the breakage of the nucleus. As we have mentioned above,the condensation of the nucleus and the DNA fragmentation are themorphological characteristics of cells under apoptosis. These resultssuggest that the conditioned medium from XC cells contain an activityinducing apoptosis in LNCAP cells. On the other hand, the conditionedmedium fails to induce apoptosis in normal human lung fibroblast (CCD 39Lu cells) and breast cancer cells (MCF-7). The nuclei of CCD 39 Lu cellsremain the same with or without incubating with the conditioned mediumof XC cells.

[0027] (b) Cell Repelling Activity

[0028] The partially purified Apogen P-1b (Q2 anionic exchangerchromatography step) isolated as described below was recently found tocontain an activity other than inducing apoptosis. We found that ApogenP-1b have the activity to repel cells away. This activity is opposite tothat of growth factors; many growth factors such as Platelet DerivedGrowth Factor (PDGF), Epidermal Growth factor (EGF), Fibroblast Growthfactor (FGF) or Transforming Growth factor (TGF) function as a“chemoattractant”—which means that these growth factors attract cellstoward them. (Grotendorst, G. R. et al., Proc. Natl. Acad. Sci. 78:3669,1981; Grant, M. B. et al Invest. Ophthal. Visual Science. 33:3292,1992). This finding suggests that Apogen P-1b isolated in this inventionplays an opposite biological function as that of a growth factor. Forexample, growth factors induce cell growth and attract cells, whereasApogen P-1b induces cell death and repels cells. Apogen P-1b is thefirst “chemorepellent” found in the field of modern biology.

[0029] A tissue culture device called Transwell Insert purchased fromCostar (Cambridge, Mass.) was used to discover the chemorepellentactivity of Apogen P-1b. This device, which has been widely used for thestudies of cell migration/invasion, contains an upper chamber and alower chamber. Between these two chambers is a polyester microporousmembrane with 3.0 μm pore size, which allows cells to migrate throughthe membrane. Tested cells are grown on the upper chamber, and thetested compound is placed in the lower chamber. If this tested compoundis a chemoattractant, we should see more cells migrate through themembrane than the control sample. In our experiments, Hep G2 (100,000cells) cells, which have a cell size 3-4 times as big as the membranepore size, were grown in the upper chamber for 2 hours, and then, thepartially purified Apogen-1b (30 μl) isolated by ammonium sulfateprecipitation and Q2 HPLC chromatography as described above was placedin the lower chamber. After 15 hours, cells that have migrated throughthe membrane were collected by treating the membrane with 0.2 ml oftrypsin solution for 30 min. Cells in ten microliters of the trypsinsolution were counted in a hemacytometer. In several experiments, wefound that the partially purified Apogen-1b contained an activitydecreasing the number of cells going through the membrane. For example,in one experiment, in the presence of the partially purified ApogenP-1b, the cell number in 10 microliters trypsin solution (which are thecells go through membrane) is 24+-4, whereas the number of cells that gothrough the membrane in the control experiment is 82+-27. This resultsuggests that the partially purified Apogen P-1b prevents Hep G2 cellsfrom migrating through membrane. To unequivocally show that Apogen P-1brepel cells, an inverted experiment was installed, instead of placingApogen P-1b in the lower chamber, we placed Apogen P-1b in the upperchamber, after 12 hours, we found that 56+-19 cells went throughmembrane compared with control experiment of 30+-1.7 cells per 10microliters of trypsin solution. The statistically significant increaseor decrease in the number of cells going through the membrane byalternatively placing Apogen P-1b in the upper or lower chamber of thistissue culture device strongly suggests that Apogen P-1b repels cells.

[0030] (3) Isolation of Apogen P-1 from XC Conditioned Medium

[0031] The Apogen P-1 present in the conditioned medium was isolated bythe following steps:

[0032] Step 1: Ammonium Sulfate Precipitation

[0033] Apogen P-1 was precipitated by 80% saturated of ammonium sulfateby adding 561 g of ammonium sulfate per liter of conditioned medium.Pellet was collected by centrifugation and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4). After removal of ammonium sulfate bydialysis, the dissolved proteins were separated by a Q2 HPLC column.

[0034] Step 2: Q2 HPLC Chromatography

[0035] The dissolved proteins isolated by ammonium sulfate precipitationwere concentrated and loaded on to a Q2 column (Bio-Rad) which wasfurther developed by a linear gradient constructed by buffer A (10 mMTris-HCl, pH 7.4) and buffer B (10 mM Tris-HCl, pH 7.4, 0.55 M NaCl)using BioRad's BioLogic HPLC system. The linear gradient was constructedby increasing buffer B from 0% to 100% in buffer A within 10 min (20milliliter elution volume and thereafter the column was eluted with 100%buffer B for 5 min.

[0036] The Apogen P-1 activity was assayed by the induction of apoptosisin LNCAP cells. We found that there were three activity peaks across thechromatogram profile. Fraction 5 to 7 caused 70% cell death; fraction8-10 caused 65% cell death; and fraction 11-14 caused 90% cell death in18 hr. We collected fractions 5-7 and named it Apogen P-1a; fractions8-10 was named Apogen P-1b; and fractions 11 to 14 was named ApogenP-1c. These three Apogen P-1's were further purified by a reverse phasecolumn.

[0037] Step 3: Reverse Phase Chromatography.

[0038] Apogen P-1a, Apogen P-1b and Apogen P-1c were separatelyconcentrated to 1.5 ml. One ml of methanol containing 0.05%trifluoracetic acid was added. In each sample, a large amount of proteinwas precipitated by this treatment. Whereas, the apoptosis inducingactivity remained in the supernatant. The supernatant was then appliedto a reverse phase RP-4 column (Micra Scientific Inc) and developed by alinear gradient constructed by solution A (H20, 0.05% TFA) and solutionB (Methanol, 0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A within 10 min (20milliliter elution volume and thereafter the column was eluted with 100%solution B for 5 min).

[0039] Step 4: Preparative Electrophoresis.

[0040] Apogen 1c isolated by anion exchange chromatography was purifiedby both Reverse phase chromatography (step 3) and PreparativeElectrophoresis by a MiniPrep Gel electrophoresis (Bio-Rad). In thereverse phase chromatogram of Apogen P-1a, fractions 12-13 had activityinducing 80% cell death in LNCAP cells at 10 hr. In the reverse phasechromatogram of Apogen P-1b, fractions 14 and 15 had activity inducing45% cell death in LNCAP cells at 18 hr.

[0041] In the reverse phase chromatogram of Apogen P-1c, fraction No. 5had activity inducing 52% cell death in LNCAP cells at 18 hr.

[0042] The purity of the isolated Apogen P-1a, Apogen P-1b and ApogenP-1c was checked with SDS-polyacrylamide gel electrophoresis stainedwith silver staining.

[0043] (1) Apogen P-1a: a protein band with molecular weight of 70 KDwas obtained. This result suggests the nearly successful purification ofApogen P-1a, which has the molecular weight of 70 KD on SDS-PAGE.

[0044] (2) Apogen P-1b: A single faint protein band with molecularweight of 55 KD was obtained. This result suggests the successfulpurification of Apogen P-1b, which has the molecular weight of 55 KD onSDS-PAGE.

[0045] (3) Apogen P-1c: The purification of Apogen 1c by Reverse Phasechromatography leads to the isolation of a 70 KD protein whereas thepurification of Apogen-1c by preparative electrophoresis leads to thepurification of a 57 KD protein. A major protein band with molecularweight of 70 KD was obtained by Reverse Phase chromatography. A 57 KDprotein, on the other hand, was isolated by preparative electrophoresis.

[0046] Our next step, obviously, will be put our entire efforts onobtaining enough of the protein band for an amino acid sequence.

(B) Isolation of Apogen P-2

[0047] (1) Source of Apogen P-2

[0048] Apogen P-2 was isolated from the conditioned medium of a cellline called C3H 10T1/2, which was derived from mouse embryo cells (ATCCCCL 226). C3H 10T1/2 cells were first grown in alpha Modification ofEagle's Medium (alpha-MEM) containing 10% Fetal bovine serum (FBS) for 3days. Cells were then washed with PBS (3×100 ml) to remove serum andthen grown in alpha-MEM containing no FBS for 4 days. From this serumfree conditioned medium, we detected an activity inducing apoptosis in aprostate cancer cell line called LNCAP. On the other hand, normal humanlung fibroblast cell line (CCD 39 Lu) was not affected by this activity.

[0049] (2) Activity of Apogen P-2

[0050] (a) Apoptosis Inducing Activity

[0051] The activity of the crude conditioned medium of C3H 10T1/2 cellswas tested on the following cell lines: LNCAP (Prostate cancer), breastcancer cells (MCF-7), and CCD 39 Lu (normal lung fibroblast). When the10-fold concentrated conditioned medium was incubated for 18 hours withthe above cell lines in the presence of 5% serum, the conditioned mediuminduced apoptosis in LNCaP(100%) and without activity in CCD 39 Lu (0%).To morphologically demonstrate that the C3H 10TI/2 conditioned mediumcontains activity inducing apoptosis, LNCAP cells were incubated withcontrol medium or with the conditioned medium treated as described asabove for 15 hr and then stained with Hoechst dye for 2 hours. Thenuclei of the LNCAP cells that have been incubated with control mediumare normal and healthy. However, the nuclei of the LNCAP cells that havebeen incubated with the conditioned medium show the characteristic ofapoptosis. First, the conditioned medium causes the condensation of thenucleus, demonstrated by the more intense fluorescent light as comparedwith the control nucleus. Secondly, the condensation of the nucleus isaccompanied by the fragmentation of DNA, demonstrated by the breakage ofnucleus. As we have mentioned above, the condensation of the nucleus andDNA fragmentation are the morphological characteristics of cells underapoptosis. The same holds true of breast cancer cells (MCF-7) in which85% apoptotic effect was observed after 18 hours of exposure to P-2.These results suggest that the conditioned medium from C3H10T1/2 cellscontains an activity inducing apoptosis in LNCAP and MCF-7 cells. On theother hand, the conditioned medium fails to induce apoptosis in normalhuman lung fibroblast (CCD 39 Lu cells). The nuclei of CCD 39 Lu cellsremain the same with or without ncubating with the conditioned medium ofC3H10T1/2 cells.

[0052] (b) Cell Repelling Activity

[0053] The partially purified Apogen P-2, isolated by ammonium sulfateprecipitation, hydroxylapatite, and heparin treatment as describedabove, was recently found to contain an activity other than inducingapoptosis. Similar to Apogen P-1b, Apogen P-2 has the activity to repelcells away. Transwell Insert purchased from Costar (Cambridge, Mass.)was used to discover the chemorepellent activity of Apogen P-2. Thisdevice, which has been widely used for the study of cellmigration/invasion, contains an upper chamber and a lower chamber.Between these two chambers is a polyester microporous membrane with 3.0μm pore size, which allows the cells to migrate through the membrane.The tested cells (HL-60) were grown on the upper chamber, and the testedcompound (Apogen P-2) was placed in the lower chamber. In ourexperiments, HL-60 (100,000 cells) cells, which have a cell size 2-3times as big as the membrane pore size, were grown in the upper chamberfor 2 hours, and then, the partially purified Apogen P-2 (30 μl)isolated by ammonium sulfate precipitation, hydroxylapatite, and Heparinagarose as described above was placed in the lower chamber. After 6hours, cells that have migrated through the membrane were collected fromthe lower chamber; the medium in lower chamber (0.6 ml) was centrifugedfor 10 min; and the HL-60 cells that went through the membrane werecollected and resuspended in 80 μl of PBS. The cells in ten microlitersof the PBS solution were counted in a hemacytometer. In severalexperiments, we found that the partially purified Apogen P-2 containedan activity that decreases the number of cells going through themembrane. For example, in one experiment, in the presence of thepartially purified Apogen P-2, the number of cells in 10 microliters PBSsolution (which are the cells go through the membrane) is 47+-5.6,whereas the number of cells that go through the membrane in the controlexperiment is 213+-40. At this moment, no apoptosis was observed inHL-60 cells present in the upper chamber. This result suggests that thepartially purified Apogen P-2 prevents the HL-60 cells from migratingthrough membrane.

[0054] (3) Isolation of Apogen P-2 from C3H10T1/2 Conditioned Medium

[0055] The Apogen P-2 present in the conditioned medium was isolated bythe following steps:

[0056] Step 1: Ammonium Sulfate Precipitation.

[0057] Apogen P-2 was precipitated by ammonium sulfate (80% saturated)by adding 561 g of ammonium sulfate per liter of conditioned medium. Thepellet was collected by centrifugation, and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4).

[0058] Step 2: Hydroxylapatite Treatment.

[0059] After removal of ammonium sulfate by dialysis in 10 mM Tris-HCl(pH 7.5), the dissolved proteins were incubated with Hydroxylapatite gel(Bio-Gel HTP gel, Bio-Rad) for 1 hr. After removing HTP gel bycentrifugation, the activity inducing apoptosis in LNCAP cells was foundto be present in the supernatant, which was then further treated withHeparin agarose gel.

[0060] Step 3: Heparin Agarose Treatment.

[0061] The supernatant from step 2 was further incubated with Heparinagarose (Sigma) for 1 Hr. After removing HTP gel by centrifugation, theactivity inducing apoptosis in LNCAP cells was found to be present inthe supernatant.

[0062] Step 4: Reverse Phase Chromatography.

[0063] Apogen P-2 present in the supernatant of Heparin agarose in step3 was further purified by reverse phase chromatography. Apogen P-2 wasconcentrated to 1 ml. One milliliter of methanol containing 0.05%Trifluoracetic acid was added. A large amount of protein wasprecipitated by this treatment. Whereas, the apoptosis inducing activity(P-2) remained in the supernatant. The supernatant was then applied to areverse phase RP-4 column (Micra Scientific Inc) and developed by alinear gradient constructed by solution A (H20, 0.05% TFA) and solutionB (Methanol, 0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A in 10 min (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min.

[0064] In the reverse phase chromatogram of Apogen P-2, fractions 12-14have activity inducing 80% cell death in LNCAP cells at 12 hr. Thepurity of the isolated Apogen P-2 was checked with SDS-polyacrylamidegel electrophoresis stained with silver staining, and a single proteinband with molecular weight of 65 Kd was obtained.

(C) Isolation of Apogen L

[0065] (1) Source of Apogen L

[0066] Apogen L was isolated from the conditioned medium of XC cell line(ATCC CCL 165). XC cells were grown in Dulbecco's Modification ofEagle's Medium (DMEM) containing 10% Fetal bovine serum (FBS) for 4days. From this conditioned medium, we detected an activity inducingapoptosis in a leukemia cell line called HL-60. On the other hand,normal human lung fibroblast cell line (CCD 39 Lu) is not affected bythis activity.

[0067] (2) Isolation of Apogen L from XC Conditioned Medium

[0068] The Apogen L present in the conditioned medium was isolated bythe following steps:

[0069] Step 1: DE52 Absorption

[0070] The conditioned medium was incubated with the anion exchanger, DE52 (Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubationmixture was centrifuged and DE 52, which binds Apogen L was collectedand washed with 10 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. Apogen Lwas then eluted from DE 52 cellulose by 10 mM Tris-HCl (pH 7.5)containing 0.5 M NaCl.

[0071] Step 2: Heparin Agarose Absorption

[0072] Apogen L isolated as described in step 1 was further absorbed byHeparin agarose (Sigma) by incubating Apogen L with Heparin agarose for1 hr. Heparin agarose was collected by centrifugation and was washedwith 10 mM Tris-HCl (pH 7.5). Apogen L absorbed in Heparin agarose wasthen eluted by 2 M NaCl.

[0073] Step 3: Q2 HPLC Chromatography

[0074] Apogen L isolated as described above was concentrated and loadedonto a Q2 column (Bio Rad) which is further developed by a lineargradient constructed by buffer A (10 mM Tris-HCl, pH 7.4) and buffer B(10 mM Tris-HCl, pH 7.4, 0.5 M NaCl) using Bio-Rad's BioLogic HPLCsystem. The linear gradient was constructed by increasing buffer B from0% to 100% in buffer A within 10 min. The purity of the isolated ApogenL was checked with SDS-polyacrylamide gel electrophoresis stained withsilver staining. A single protein band with molecular weight of 55 Kdwas obtained.

[0075] (3) Activity of Apogen L

[0076] The activity of Apogen L isolated as described above was testedon the following cell lines: HL-60 (leukemia) and CCD 39 Lu (normal lungfibroblast). To morphologically demonstrate that Apogen L containsactivity inducing apoptosis, HL-60 cells were incubated with Apogen Lisolated as described as above for 15 hr and then stained with Hoechstdye for 2 hours. The nuclei of the HL-60 cells that have been incubatedwith control medium are normal and healthy. However, the nuclei of theHL-60 cells that have been incubated with Apogen L show thecharacteristic of apoptosis. First, Apogen L causes the condensation ofnucleus, demonstrated by the more intense fluorescent light comparedwith the control nucleus. Secondly, the nucleus condensation isaccompanied by the fragmentation of DNA, demonstrated by the breakage ofnucleus. As we have mentioned above, the nucleus condensation and DNAfragmentation are the two morphological characteristics of cells underapoptosis. These results suggest that the isolated Apogen L contains anactivity inducing apoptosis in HL-60 cells. Apogen L also inducesapoptosis in MCF-7 (breast cancer) cells. On the other hand, theconditioned medium fails to induce apoptosis in normal human lungfibroblast (CCD 39 Lu cells).

EXAMPLES A. Methods

[0077] 1. Preparation of Condition Media.

[0078] A. Preparation of XC Condition Medium for Isolation of ApogenP-1.

[0079] Apogen P-1 was isolated from the conditioned medium of a cellline called XC, which was derived from a rat tumor (ATCC CCL 165). XCcells were first seeded in a roller bottle (Polystyrene, areasurface=850 Cm2, Corning) in Dulbecco's Modification of Eagle's Medium(DMEM) containing C02, 10% fetal bovine serum (FBS), non-essential aminoacids, penicillin and streptomycin for 3 days. XC cells were then washedwith PBS (3×100 ml) to remove serum and then grown in 100 ml of DMEMcontaining no FBS (with C02), non-essential amino acids, penicillin andstreptomycin) for 4 days. The conditioned medium was collected andclarified by centrifugation.

[0080] B. Preparation of C3H 10T1/2 Condition Medium for Isolation ofApogen P-2 .

[0081] Apogen P-2 was isolated from the conditioned medium of a cellline called C3H10T1/2, which was derived from a mouse embryo and waspurchased from American Type Culture Collection (ATCC CCL 226).C3H10T1/2 cells were first seeded in a roller bottle (Polystyrene, areasurface=850 Cm2, Corning) in alpha Modification of Eagle's Medium(alpha-MEM) containing C02, 10% Fetal bovine serum (FBS), penicillin andstreptomycin for 3 days. C3H10T1/2 cells were then washed with PBS(3×100 ml) to remove serum and then grown in 100 ml of alpha MEMcontaining no FBS (with C02, penicillin and streptomycin) for 4 days.The conditioned medium was collected and clarified by centrifugation.

[0082] C. Preparation of XC Condition Medium for Isolation of Aooaen L.

[0083] Apogen L was isolated from the conditioned medium of a cell linecalled XC, which was derived from rat tumor (ATCC CCL 165). XC cellswere first seeded in a roller bottle (Polystyrene, area surface=850 Cm2,Corning) in Dulbecco's Modification of Eagle's Medium (DMEM) containingpenicillin, streptomycin, C02, non-essential amino acids and 10% Fetalbovine serum (FBS) for 4 days. The conditioned medium was collected andclarified by centrifugation.

[0084] 2. Assays

[0085] (a) Cell Death (Apoptosis) Assay

[0086] Prostate cancer cell line LNCAP was routinely used for theisolation of Apogen P-1 and Apogen P-2, whereas leukemia cell line HL-60was used for the isolation of Apogen L. The methods of assays were asfollows: LNCAP or HL-60 (1,000 cells) was seeded in 10 microliters RPMIcontaining 15% or 20% Fetal bovine serum, penicillin and streptomycin at37 degrees, 5% C02 in Microtray plates (25 μl wells, Robbins ScientificCorp.). The tested sample (10 μl) was added 3-4 hours after cells wereseeded. After incubation of the tested sample with cells for 15 hours,two microliters of Hoechst dye (0.03 ng/ml in PBS) was added. Two hourslater, cells that were stained with Hoechst dye were examined underfluorescence microscope. The nuclei of apoptotic cells showed DNAcondensation and fragmentation, which are easily identified by Hoechstdye staining. The percentage of apoptotic cells was calculated by thefollowing equation:${\% \quad {Apoptotic}\quad {c{ells}}} = \frac{\begin{matrix}{{Number}\quad {of}\quad {cells}\quad {with}\quad {DNA}} \\{{condensation}\quad {and}\quad {fragmentation}}\end{matrix}}{{Total}\quad {cell}\quad {number}}$

[0087] (b) Cell Repelling Assay

[0088] There are two reasons that Hep G2 cells are chosen for the studyof cell repelling activity. First, Hep G2 cells are not sensitive toApogen P-1 in inducing apoptosis. Secondly, the cell size of Hep G2 cellis about 3-4 times as big as the pore size of the membrane on theTranswell Insert, which is a good cell size for cell migration/invasionstudy. A tissue culture device called Transwell Insert purchased fromCostar (Cambridge, Mass.) was used to discover the chemorepellentactivity of Apogen P-1b. This device, which has been widely used for thestudies of cell migration/invasion, contains an upper chamber and alower chamber. Between these two chambers is a polyester microporousmembrane with 3.0 μm pore size, which allows cells to migrate throughthe membrane. Tested cells were grown on the upper chamber, and thetested compound was placed in the lower chamber. If this tested compoundis a chemoattractant, we should see more cells migrate through membranethan the control sample. In our experiments, Hep G2 (100,000 cells)cells, which have a cell size 3-4 times as big as the membrane pore sizewere grown in the upper chamber (Minimum Essential Medium Eaglecontaining 10% FBS, PS and nonessential amino acid, 0.1 ml) for 2 hours,and then the partially purified Apogen-lb (30 μl) isolated by ammoniumsulfate precipitation and Q2 HPLC chromatography as described above wasplaced in the lower chamber which contains 0.6 ml of the same growthmedium for Hep G2 cells. After 15 hours, cells that have migratedthrough the membrane were collected by treating the membrane with 0.2 mlof trypsin solution for 30 min. Cells in ten microliters of the trypsinsolution were counted in a hemacytometer.

[0089] 3. Protein Isolation

[0090] A. Isolation of Apogen P-1

[0091] Step 1: Ammonium Sulfate Precipitation

[0092] Apogen P-1 was precipitated by 80% saturated of ammonium sulfateby adding 561 g of ammonium sulfate per liter of XC conditioned medium.The pellet was collected by centrifugation, and the proteins weredissolved in 10 mM Tris-HCl (pH 7.4). After removal of ammonium sulfateby dialysis, the dissolved proteins were separated by a Q2 HPLC column.

[0093] Step 2: Q2 HPLC Chromatography

[0094] The dissolved proteins isolated by ammonium sulfate precipitationwere concentrated and loaded onto a Q2 column (Bio Rad) which wasfurther developed by a linear gradient constructed by buffer A (10 mMTris-HCl, pH 7.4) and buffer B (10 mM Tris-HCl, pH 7.4, 0.55 M NaCl)using BioRad's BioLogic HPLC system. The linear gradient was constructedby increasing buffer B from 0% to 100% in buffer A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% buffer B for 5 min. The Apogen P-1 activity was assayed by theinduction of apoptosis in LNCAP cells. We found that there are threeactivity peaks across the chromatogram profile. Fractions 5 to 7 cause70% cell death; fractions 8-10 cause 65% cell death; and fractions 11-14caused 90% cell death in 18 hr. We collected fractions 5-7 and named itApogen P-1a; fractions 8-10 was named Apogen P-1b; and fractions 11-14was named Apogen P-1c. These three Apogen P-1's were further purified bya reverse phase column.

[0095] Step 3: Reverse Phase Chromatography.

[0096] Apogen P-1a, Apogen P-1b and Apogen P-1c were separatelyconcentrated to 1.5 ml. One ml of methanol containing 0.05%Trifluoracetic acid was added. In each sample, a large amount of proteinwas precipitated by this treatment. Whereas, the apoptosis inducingactivity remained in the supernatant. The supernatant was then appliedto a reverse phase RP-4 column (Micra Scientific Inc) and developed by alinear gradient constructed by solution A (H20, 0.05% TFA) and solutionB Methanol, 0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A within 10 min., 20milliliter elution volume, and thereafter the column was eluted with100% solution B for 5 min.

[0097] Step 4: Preparative Electrophoresis

[0098] Apogen 1c isolated by anion exchange chromatography was purifiedby both Reverse Phase Chromatography (step 3) and PreparativeElectrophoresis by a MiniPrep Gel electrophoresis (Bio-Rad). In thereverse phase chromatogram of Apogen P-1a, fractions 12-13 have activityinducing 80% cell death in LNCAP cells at 10 hr.

[0099] In the reverse phase chromatogram of Apogen P-1b, fractions 14and 15 have activity inducing 45% cell death in LNCAP cells at 18 hr.

[0100] In the reverse phase chromatogram of Apogen P-1c, fraction No. 5has activity inducing 52% cell death in LNCAP cells at 18 hr.

[0101] The purity of the isolated Apogen P-1a, Apogen P-1b and ApogenP-1c were checked with SDS-polyacrylamide gel electrophoresis stainedwith silver staining.

[0102] (1) Apogen P-1a: A protein band with molecular weight of 70 KDwas obtained. This result suggests the nearly successful purification ofApogen P-1a, which has a molecular weight of 70 KD on SDS-PAGE.

[0103] (2) Apogen P-1b: A single faint protein band with molecularweight of 55 KD was obtained. This result suggests the successfulpurification of Apogen P-1b, which has a molecular weight of 55 KD onSDS-PAGE.

[0104] (3) Apogen P-1c: The purification of Apogen 1c by Reverse Phasechromatography leads to the Isolation of a 70 KD protein, whereas thepurification of Apogen 1c by preparative electrophoresis leads to thepurification of a 57 KD protein. A major protein band with molecularweight of 70 KD was obtained by Reverse Phase chromatography. A 57 KDprotein, on the other hand, was isolated by preparative electrophoresis.

[0105] B. Isolation of Apogen P-2

[0106] The Apogen P-2 present in C3H10T1/2 conditioned medium wasisolated by the following steps:

[0107] Step 1: Ammonium Sulfate Precipitation.

[0108] Apogen P-2 was precipitated by ammonium sulfate (80% saturated)by adding 561 g of ammonium sulfate per liter of conditioned medium. Thepellet was collected by centrifugation, and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4).

[0109] Step 2: Hydroxylapatite Treatment.

[0110] After removal of ammonium sulfate by dialysis in 10 mM Tris-HCl(pH 7.5), the dissolved proteins were incubated with hydroxylapatite gel(Bio-Gel HTP gel, Bio-Rad) for 1 hr. After removing HTP gel bycentrifugation, the activity inducing apoptosis in LNCAP cells was foundto be present in the supernatant, which was then further treated withHeparin agarose gel.

[0111] Step 3: Heparin Agarose Treatment.

[0112] The supernatant from step 2 was further incubated with Heparinagarose (Sigma) for 1 hr. After removing HTP gel by centrifugation, theactivity inducing apoptosis in LNCAP cells was found to be present inthe supernatant.

[0113] Step 4: Reverse Phase Chromatography.

[0114] Apogen P-2 presents in the supernatant of Heparin agarose in step3 was further purified by reverse phase chromatography. Apogen P-2 wasconcentrated to 1 ml. One milliliter of methanol containing 0.05%trifluoacetic acid was added. Large amounts of proteins wereprecipitated by this treatment. Whereas, the apoptosis inducing activity(P-2) remained in the supernatant. The supernatant was then applied to areverse phase RP-4 column (Micra Scientific Inc.) and developed by alinear gradient constructed by solution A (H20, 0.05% TFA) and solutionB Methanol (0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min. In the reverse phase chromatogram of ApogenP-2, fractions 12-14 have activity inducing 80% cell death in LNCAPcells at 12 hr. The purity of the isolated Apogen P-2 was checked withSDS-polyacrylamide gel electrophoresis stained with silver staining. Asingle protein band with molecular weight of 65 Kd was obtained.

[0115] C. Isolation of Apogen L

[0116] The Apogen L present in the conditioned medium was isolated bythe following steps:

[0117] Step 1: DE52 Absorption

[0118] The conditioned medium was incubated with the anion exchanger, DE52 (Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubationmixture was centrifuged, and DE 5 2, which binds Apogen L, was collectedand washed with 10 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. Apogen Lwas then eluted from DE 5 2 cellulose by 10 mM Tris-HCl (pH 7.5)containing 0.5 M NaCl.

[0119] Step 2: Heparin Agarose Absorption

[0120] Apogen L isolated as described in step 1 was further absorbed byHeparin agarose (Sigma) by incubating Apogen L with Heparin agarose for1 hr. Heparin agarose was collected by centrifugation and was washedwith 10 mM Tris-HCl (pH 7.5). Apogen L absorbed in Heparin agarose wasthen eluted by 2 M NaCl.

[0121] Step 3: Q2 HPLC Chromatography

[0122] Apogen L isolated as described above was concentrated and loadedonto a Q2 column (Bio Rad) which was further developed by a lineargradient constructed by buffer A (10 mM Tris-HCl, pH 7.4) and buffer B(10 mM 22 Tris-HCl, pH 7.4, 0.5 M NaCl) using Bio-Rad's BioLogic HPLCsystem. The linear gradient was constructed by increasing buffer B from0% to 100% in buffer A in 10 min. The purity of the isolated Apogen Lwas checked with SDS-polyacrylamide gel electrophoresis stained withsilver staining. A single protein band having activity with a molecularweight of approximately 55 Kd was obtained.

[0123] 4. Isolation of Bovine Fetuin as a Component of Protein P-2 andthe Apoptotic Effect Thereof in Tumor Cell Lines.

[0124] The observation that Apogen P-1a, P-1b, P-1c, P-2 and L wereisolated from embryonic cell lines led us to speculate that newborn orembryonic tissue may secrete “Apogen,” which may selectively induceapoptosis in tumor cell lines. Thus, due to this speculation, a proteinnamed “Fetuin” has raised our attention for the following reasons: (1)Fetuin is mainly a fetal protein, in the sense that the highestconcentrations are found in serum and body fluids of embryos andfetuses. For example, the concentration of fetuin in bovine serumdrastically decreases, probably within a few days after birth, to 1-2%of the fetal level. (Yang, et al., Biochim. Biophy. Acta. 1130, 149-1561992). (2) A histochemical study has shown that fetuin may controltissue remodelling and physiological cell death during embryonicdevelopment. (Von Bulow, et al., Histochemistry 99:13-22, 1993). Thisresult raises the possibility that fetuin may contain activity inducingcell death (apoptosis).

[0125] Additionally, a protein with an amino acid sequence identical toFetuin was isolated from the preparation of Apogen P-2. Thus, thecomposition of Apogen P-2 consists at least in part of fetuin.

[0126] We therefore prepare and obtain fetuin and test fetuin in ourapoptosis assay. Interestingly, we found that only bovine fetuin that isprepared by a special method is able to induce apoptosis in tumor celllines. The commercial fetuin that is prepared by ammonium sulfateprecipitation and EDTA treatment was found to contain a very lowactivity in inducing apoptosis in tumor cells.

[0127] 4A. Preparation of Bovine Fetuin.

[0128] Bovine fetuin was prepared by the modified Spiro method (Spiro R.G., Journal of Biological Chemistry 235, 10: 2860, 1960) according tothe following steps:

[0129] 1. One hundred milliliters of Fetal Bovine Serum (FBS).

[0130] 2. Add two hundred milliliters of 0.05 M Zinc Acetate containing30% (V/V) ethanol, adjust to pH 6.4 by 1M NH40H-NH4Cl, let stand 15hours at −5° C.

[0131] 3. Collect the supernatant by centrifugation, add 1.0 M BariumAcetate and 95% ethanol to give 0.03 M Barium Acetate, 25% ethanol. Letstand 2 hours at −5° C.

[0132] 4. Collect the supernatant by centrifugation, add 95% ethanol togive 40% ethanol. Let stand 15 hours at −10° C.

[0133] 5. Collect the precipitate. Dissolve the pellet by phosphatebuffer saline.

[0134] The purified fetuin showed a single protein band with apparentmolecular weight of 63 Kd on SDS-PAGE.

[0135] 4B. Induction of Apoptosis in Tumor Cell Lines Using BovineFetuin.

[0136] Fetuin purified from fetal bovine serum by the proceduredescribed above was dissolved in phosphate buffer saline (PBS). The freeZinc Acetate and Barium Acetate were removed by repetitiveconcentration. Fetuin was tested in LNCaP and HL-60 cells. LNCaP orHL-60 (1,000 cells) was seeded 10 microliters RPMI containing 15% or 20%Fetal bovine serum, penicillin and streptomycin at 37 degree, 5% CO2 inmicrotray plates (25 μl wells, Robbins Scientific Corp.). Fetuin (in 10μl PBS) at concentration of 100 ng/ml was added 3-4 hours after cellswere seeded. After incubation of the tested sample with cells for 15hours, two microliters of Hoechst dye (0.03 ng/ml in PBS) was added. Twohours later, cells that were stained with Hoechst dye were examinedunder fluorescence microscope. The nuclei of apoptotic cells showed DNAcondensation and fragmentation, which can be easily identified byHoechst dye staining. The percentage of apoptotic cells was calculatedby the following equation:${\% \quad {Apoptotic}\quad {c{ells}}} = \frac{\begin{matrix}{{Number}\quad {of}\quad {cells}\quad {with}\quad {DNA}} \\{{condensation}\quad {and}\quad {fragmentation}}\end{matrix}}{{Total}\quad {cell}\quad {number}}$

[0137] The nuclei of the LNCaP cells that have been incubated withcontrol sample (PBS) are normal and healthy. However, the nuclei of theLNCaP cells that have been incubated with fetuin (100 ng/ml in PBS) showthe characteristics of apoptosis. First, the cells in the presence offetuin showed the condensation of nucleus, demonstrated by the moreintense fluorescent light compared with the control nucleus. Secondly,the nucleus condensation is accompanied by the fragmentation of DNA,demonstrated by the breakage of nucleus. As the condensation of thenucleus and the DNA fragmentation are the two morphologicalcharacteristics of cells under apoptosis. These results suggest thatfetuin contains an activity inducing apoptosis in LNCaP cells. Thenuclei of the HL-60 cells that have been incubated with control buffer(PBS) are normal and healthy. However, the nuclei of the HL-60 cellsthat have been incubated with fetuin show the characteristics ofapoptosis. Fetuin causes the condensation of nucleus, demonstrated bythe more intense fluorescent light compared with the control nucleus.Secondly, the nucleus condensation is accompanied by the fragmentationof DNA, demonstrated by the breakage of the nucleus. As we havementioned above, the nucleus condensation and DNA fragmentation are thetwo morphological characteristics of cells under apoptosis. Theseresults suggest that fetuin contains an activity inducing apoptosis inHL-60 cells.

[0138] 4C. Bovine Fetuin Selectively Induces Apoptosis in Cancer CellsWithout Having an Effect on Normal Cell Lines.

[0139] We compared the effect of fetuin on the induction of apoptosis invarious cell lines. At a concentration of 50 μg/ml, fetuin prepared asdescribed above strongly induced apoptosis in tumor cell lines such as:LNCaP (prostate cancer), PC-3 (prostate cancer), HL-60 (leukemia), MCF-7(breast cancer), Colo 205 (colon cancer), Calu-1 (lung cancer). Normallung fibroblast (CCD 39 Lu), on the other hand, is not affected byfetuin.

[0140] Fetuin was found to be inactive in inducing apoptosis in CCD 39Lu cells (normal lung fibroblast) at the concentration (25 μg/ml) thathighly induced apoptosis in LNCaP (prostate cancer) or HL-60 cells(leukemia). Fetuin (25 μg/ml) prepared as described above was incubatedwith CCD 39 Lu cells grown in MEM in a microtray plate for 15 hours. TheCCD 39 Lu cells remained morphologically unchanged in the presence offetuin. At this concentration (25 μg/ml) of fetuin and in the presenceof fetuin, less MCF-7 cells remaining, due to cell death and cellshrinkage, was observed.

[0141] 4D. Only Fetuin Prepared by the Method Described Above is Able toInduce Apoptosis in Tumor Cell Lines.

[0142] We found that fetuin purchased from Sigma has a very low activityin inducing apoptosis in LNCaP cells. However, fetuin (25 μg/ml)prepared in our laboratory by the method described in Section 4A aboveinduces apoptosis in LNCaP cells by up to 90% in 4 hours. For the fetuinpurchased from Sigma, apoptosis inducing activity was observed only at avery high concentration (>250 μg/ml) and at long incubation time (2days). We estimated that the activity of fetuin prepared in ourlaboratory is more than fifty thousand folds higher than that of fetuinprepared by other methods.

[0143] In the years of research following the original findings, I haveobserved that fetuin from Sigma induces apoptosis at a very highconcentration and at a long incubation time (2 days). It isconservatively estimated that the fetuin as prepared in Section 4A hasmore than one hundred times greater apoptotic activity than fetuinprepared by other methods. While this is not as dramatic as the 50,000times increase as originally reported, it still represents a significantapoptotic advantage over previously available fetuin in terms ofincubation time and LD₅₀ values.

[0144] We have examined the preparation method for Sigma's fetuin andfound that these fetuins are prepared by methods including ammoniumsulfate precipitation and EDTA treatment. Both treatments may cause thedeprivation of the Zinc ion from the protein, which may cause theirreversible loss of the protein activity.

[0145] 4E. Effect of Fetal Fetuin on Leukemia Cells in vivo.

[0146] Our previous data demonstrated that fetuin induced apoptosis incancer cells in vitro. The data provided below shows that the in vivotesting of fetuin in mice having leukemia was successful. The resultsshow that fetuin has an anti-leukemia effect in mice. FIG. 1 shows theincrease in survival of leukemia-bearing mice treated with fetal fetuin.

Method

[0147] Forty DBA/2 female mice (17-20 grams; Simonsen Laboratories,Inc., Gilroy, Calif.) kept on a standard diet and water ad libitum wereinoculated with tumor cell line P388D1 (ATCC cell line number CCL46).The mice were randomly segregated into groups of ten (10). Zinc-chargedfetal fetuin (10 mg/ml) were intraperitoneally injected into group I at0.002 ml/mouse, group II at 0.02 ml/mouse and group III at 0.2 ml/mouse.Group IV was the control group, which was injected with 0.5 ml of salinesolution. The injections were continued for 10 days. Mortalities wererecorded for 60 days. The results were expressed as the percentageincrease in life span (ILS): ${ILS} = \frac{\begin{matrix}{{100 \times {Median}\quad {Life}\quad {Span}\quad {Treated}} -} \\{{Median}\quad {Life}\quad {Span}\quad {Controlled}}\end{matrix}}{{Life}\quad {Span}\quad {Controlled}}$

[0148]FIG. 1 shows that while 100% of the untreated leukemia-bearingmice were dead after 24 days, 80% of the mice treated with a high doseof fetuin, namely 100 mg/Kg of fetal fetuin, survived more than 58 days.This in vivo experiment demonstrates that mice bearing leukemia that aretreated with fetal fetuin have an increased life span of 141%.

[0149] 4F. Method of Preparing Supercharged Zinc Fetuin:

[0150] The method to prepare fetuin with zinc has been refined andimproved. As stated above, fetuin prepared by the method as described inSection 4A above is able to induce apoptosis in tumor cell lines.However, commercial fetuin such as from Sigma is found to have a verylow activity in inducing apoptosis in tumor cells and in inducingapoptosis in LNCaP cells. For fetuin from Sigma, apoptosis inducingactivity was observed only at a very high concentration (>250 μg/ml) andat a long incubation time (2 days), whereas fetuin (25 μg/ml) asprepared in Section 4A above induced apoptosis in LNCaP cells by up to90% in 4 hours. It was estimated that the fetuin as prepared asdescribed in Section 4A is more than fifty thousand times higher thanthat fetuin prepared by other methods. The radically different resultssuggest a fundamental difference in the chemical composition ofcommercially available fetuin and the fetuin prepared in accordance withthe procedure in Section 4A.

[0151] In the years of research following the original findings, I haveobserved that fetuin from Sigma induces apoptosis at a very highconcentration (>>5 mM) and at a long incubation time (2 days), whereasfetuin (approximately 50 μM) as prepared in Section 4A above inducedapoptosis in LNCaP cells by up to 90% in 4 hours. It is conservativelyestimated that the fetuin as prepared in Section 4A has more than onehundred times greater apoptotic activity than fetuin prepared by othermethods. While this is not as dramatic as the 50,000 times increase asoriginally reported, it still represents a significant apoptoticadvantage over previously available fetuin in terms of incubation timeand LD₅₀ values.

[0152] After looking at the methods of preparing commercially availablefetuin, it was found that ammonium sulfate precipitation and EDTAtreatment was used in preparing fetuin. It was speculated that thisammonium sulfate precipitation and EDTA treatment might cause thedeprivation of the ions from the protein to cause irreversible loss ofthe protein activity. However, it was not known whether it was the lossof zinc alone, or in combination with the loss of another ion(s), thatcaused the decreased apoptotic activity in commercially availablefetuin. While it was determined that Fetuin-Ca is inactive in inducingapoptosis (data not shown), and barium occurs in only trace amounts, todetermine which ion, or combination of ions, were most effective inincreasing the apoptotic ability of fetuin, fetuin as prepared inSection 4A above was treated with a chelating agent, such as EDTA, tostrip all the inorganic ions, including zinc, calcium, and barium fromthe protein. After removing these inorganic ions, the “naked” fetuin wastreated or incubated with 0.5 M Zinc Acetate to reload or to bind thefetuin with zinc only. The results of this refinement process are shownin examples in FIGS. 2-3 in determining the LD₅₀ value. FIGS. 2-3 showthat the improved preparation of Fetuin-Zn or “supercharged zinc fetuin”enhances fetuin's ability to induce apoptosis in cancer cells by threeto four times as compared with the original fetuin bound with zinc asprepared in Part 4A. It is hypothesized that the fetuin previously boundup with calcium and barium created an inactive form of the protein. Bystripping out all ions and replacing them with zinc, inactive fetuinmolecules were converted to active form, thereby explaining the dramaticincrease in apoptotic activity. Such supercharged zinc fetuin is avaluable step forward in the fight against cancer.

[0153] In one preferred embodiment of this preparation process:

[0154] 1. Incubation Mixture: 700 μg of fetuin (0.2 ml; as prepared bythe method as described above in Section 4A) was incubated with 0.5 mlof 0.1 EDTA for approximately one (1) hour.

[0155] 2. Concentration: Add 1.5 ml of saline solution to thisincubation mixture and concentrate to near dryness using a molecular ormolecule sieve and centrifugal force. Repeat this procedure four (4)times, so that most of the inorganic ions are removed. This “naked”fetuin will be retained on the top of the filter (molecular sieve).

[0156] 3. Incubate the “naked” fetuin (0.2 ml) with 0.5 ml of 0.5 M ZincAcetate for approximately three (3) hours.

[0157] 4. Remove the free Zinc Acetate using the combination of thesaline solution, the molecular sieve, and centrifugal force as describedin Step 2 above.

[0158] 5. A Specific Peptide Fragment from Fetuin-Zinc that CausesApoptosis in Cancer Cells.

[0159] Preparation of Fetuin Fragment:

[0160] As described above in Section 4F (Method of PreparingSupercharged Zinc Fetuin), zinc charged fetuin or supercharged zincfetuin was prepared by pre-treatment of fetal bovine fetuin with achelating agent (EDTA) to remove the inorganic ions, including zinc,calcium, and barium ions, from the fetuin. The resulting stripped fetuinwas incubated with 0.5 M Zinc Acetate in order to “supercharge”, or loadthe fetuin with zinc. Three hundred (300) micrograms of the zinc chargedfetuin was dissolved in a 50 μl saline solution and then dried in a tubeunder a vacuum. It is hypothesized that this drying step breaks apartthe zinc charged fetuin into peptide fragments.

[0161] The dried fragments (of zinc charged fetuin) of werereconstituted in 50 μl water. This fragment solution was passed througha molecular sieve membrane having a molecular weight cut-off of 10,000daltons. The resulting filtrate of fragments was collected and tested oncells in an apoptosis assay. As shown in FIG. 5, when LNCaP cells areincubated with the zinc charged fetuin fragment filtrate for six (6)hours, the LNCaP cells detach and die. Compared with the control (LNCaPwith no filtrate) as shown in FIG. 4, FIG. 5 shows that incubation ofthe prostate cancer cells with the zinc charged fetuin filtrate causesapoptosis of the cancer cells. FIG. 7 shows that the zinc charged fetuinfiltrate treated LNCaP cells also exhibit membrane “bubbling,” which isa characteristic typical of cells undergoing apoptosis. FIG. 6, whichshows LNCaP cells without the zinc charged fetuin filtrate, lacks thismembrane “bubbling” and characteristic of apoptosis.

[0162] b. Is Protease Sensitive.

[0163] Additionally, the apoptosis-inducing activity of FPF 300-09 wasfound to be protease sensitive. Incubation of the FPF 300-09 filtratewith proteinase K completely removed the apoptosis-inducing activity.Proteinase K is an enzyme that cleaves peptide bonds; chelating agentssuch as citrate and EDTA have no effect on the enzyme activity ofproteinase K.

[0164] After preparing “supercharged” zinc fetuin or zinc charged fetuinas stated in Section 4F above, the resulting composition was dried in atube and under a vacuum. This dried supercharged zinc fetuin wasreconstituted in 50 μl of water. This solution was filtered through amolecular sieve membrane (Centricon 10 tube with a molecular weightcut-off: 10,000 daltons). The filtrate was collected and treated with 5μl (1 unit/μl) proteinase K for three (3) hours at 37° C. Aftertreatment with proteinase K, the treated filtrate was filtered through amolecular sieve membrane (Centricon 10 tube) in order to remove theproteinase K. The proteinase K was retained by the membrane, and thetreated filtrate passed through the membrane.

[0165] To test the effect of a protease on the apoptotic activity of thefiltrate, the filtrate treated with proteinase K was tested on cancercells. These results were compared to the filtrate that was not treatedwith proteinase K.

[0166] The effect of proteinase K on the apoptotic ability of theFetuin-Zinc fragments is summarized in FIG. 8. Experiments 1 and 2 wereconducted with one set of zinc charged fetuin filtrate, and Experiment 3was conducted with another set of zinc charged fetuin filtrate. FIG. 8shows that incubation with a protease seems to inactivate the apoptoticeffect of the zinc charged fetuin fragment. Because a protease, such asproteinase K, cleaves peptide bonds, the test results of FIG. 8 stronglysuggest that a peptide or a protein of fetuin is responsible for theinduction of apoptosis in cancer cells.

[0167] C. The Filtrate Contains Two Malor Peptides Derived from Fetuin.

[0168] The dried and reconstituted filtrate was found to contain peptidefragments. The amino acid sequence analysis revealed two major peptidefragments in the filtrate:

[0169] (1) H-T-F-S-G-V-A-S-V-E (amino acid no. 300-309; His Thr Phe SerGly Val Ala Ser Val Glu; SEQ ID NO:1) and

[0170] (2) S-A-S-G-E-A-F-H (amino acid no. 310-317; Ser Ala Ser Gly GluAla Phe His; SEQ ID NO:2) of fetuin.

[0171] To identify which of these peptide fragments are responsible forthe apoptosis-inducing activity, the two fragments (H-T-F-S-G-V-A-S-V-E(amino acid no. 300-309; referred to herein as Fetuin Peptide Fragment(FPF 300-09); SEQ ID NO:1) and S-A-S-G-E-A-F-H (amino acid no. 310-317;SEQ ID NO:2) of the full-length fetuin molecule) were chemicallysynthesized. Upon in vitro testing of these chemically synthesizedpeptide fragments, FPF 300-09 was shown to have the greater apoptoticactivity. LNCaP (prostate cancer cells) were incubated with FPF 300-09.In FIG. 10, chemically synthesized FPF 300-09 caused membrane “bubbling”in LNCaP cells after three (3) hours of incubation. Incubation of thepeptide fragment (amino acid no. 310-317) or the peptide fragment (aminoacid no. 300-307) with LNCaP cells did not show any apoptotic activityor membrane “bubbling.” FIG. 9 shows the control of LNCaP cells withoutFPF 300-09. These results suggest that the peptide fragment that inducedapoptosis and that was present in the filtrate corresponds to amino acidno. 300-309 of the full-length fetuin.

[0172] 6. Characterization of FPF 300-09.

[0173] a. FPF 300-09 Selectively Induced Apoptosis in Cancer Cells butNot in Normal Cells.

[0174] Previously, it was found that fetuin and zinc charged fetuininduced apoptosis in various cancer cells without affecting certainnormal cells. To test whether FPF 300-09 derived from fetuin retainsthis selectivity in inducing apoptosis in cancer cells only and notaffecting normal cells, various concentrations of FPF 300-09 were testedon HT-29 (colon cancer), CCD-18 Co (normal colon), and LNCaP (prostatecancer) cells. As shown in FIG. 11, FPF 300-09 induced apoptosis inHT-29 and LNCaP cells without affecting CCD-18 Co cells. These resultssuggest that the fragment was similar to fetuin and zinc charged fetuinin selectively inducing apoptosis in cancer cells and not in normalcells.

[0175] b. FPF 300-09 Rapidly Induced Apoptosis in HT-29 (Colon Cancer)Cells.

[0176]FIG. 12 shows the effect of time on the induction of apoptosis byFPF 300-09. At four (4) hours, FPF 300-09 at a concentration of 0.4 μMinduced apoptosis in 52% of the HT-29 colon cancer cells. At six (6)hours, almost all of the HT-29 cells were induced to apoptosis. Evenwith a little more incubation time (from 4 to 6 hours), the apoptoticability of FPF 300-09 rapidly increases within this short period oftime.

[0177] c. The Peptide Fragment Derived from Fetuin is More Potent thanFetuin or Zinc Charged Fetuin in Inducing Apoptosis.

[0178] Previously, the LD₅₀ (dosage for the induction of 50% cell death)for full-length zinc charged fetuin in LNCaP cells was determined to be3-1.0 μM. However, the LD₅₀ for FPF 300-09 was determined to be 0.3-0.4μM. Hence, with a much smaller LD₅₀ value, a much smaller amount of theFPF 300-09 was required to induce the same amount of cancer cells thanis required with full-length zinc charged fetuin. Further, FPF 300-09 ismore potent in inducing apoptosis than its parent molecule (FIG. 13).

[0179] Taking into consideration the previous estimates:

[0180] (1) Fetuin as prepared in Section 4A above is approximately 100times more powerful than fetuin prepared from other methods;

[0181] (2) “Supercharged” Zinc Fetuin (zinc charged fetuin) isapproximately three to four times more powerful than the fetuin asprepared in Section 4A (see FIG. 2-3); and

[0182] (3) FPF 300-09 is approximately eight to ten times more powerfulthan “Supercharged” Zinc Fetuin (see FIG. 13),

[0183] it is estimated that FPF 300-09 is approximately several thousandtimes more powerful than fetuin prepared from other methods.

[0184] 7. Other Sequences of Fetuin from Other Sources.

[0185] In addition, peptide sequences were determined from other animalsera, including pig, sheep and mice. K. M. Dziegielewska, et. al.,Fetuin, 16-17, (R. G. Landes Co. 1995). These peptide sequences have asimilarity of 60-90% with the fetuin isolated from bovine serum. Thepresent application instructs that these similar fetuin peptidesequences will also have valuable apoptotic activity. The peptidesequences for FPF 300-09 for other species are: Human(H-T-F-M-G-V-V-S-L-G; SEQ ID NO:3) His Thr Phe Met Gly Val Val Ser LeuGly;; Pig (H-S-F-S-G-V-A-S-V-E; SEQ ID NO:4) His Ser Phe Ser Gly Val AlaSer Val Glu;; Sheep (H-T-F-S-G-V-A-S-V-E; SEQ ID NO:5) His Thr Phe SerGly Val Ala Ser Val Glu;; Rat (H-T-F-S-G-V-A-S-V-E; SEQ ID NO:6) His ThrPhe Ser Gly Val Ala Ser Val Glu;; and Mouse (H-A-F-S-P-V-A-S-V-E; SEQ IDNO:7) His Ala Phe Ser Pro Val Ala Ser Val Glu;. Id.

[0186] These analogs are also claimed as being taught by the currentapplication.

Discussion

[0187] This invention describes the methods for the isolation of fiveproteins (Apogen P-1a, Apogen P-1b, Apogen P-1c, Apogen P-2 and ApogenL) that are able to induce apoptosis in prostate cancer cells (ApogenP-1's ), in prostate cancer cells and breast cancer cells (Apogen P-2),and leukemia and breast cancer cells (Apogen L), as well as theidentification of fetuin as a component of Apogen P-2. The followingevidence leads us to believe that these apoptosis-inducing proteins arenovel and that they have never been found before: Tumor Necrosis Factor(TNF), Transforming Growth Factor (TGF-Beta), Fas ligand and TRAIL arethe proteins reported to induce apoptosis in certain cell lines. (Lin,J. K. et al., Cancer Research 52:385, 1992. Kawakawi, et al., J. ofCellular Physiology 138:1, 1989; Wiley, S. R. et al., Immunity 3:673,1995; Krammer, et al. “Apoptosis in the APO-1 System”, Apoptosis: Themolecular Basis of Cell Death, Cold Spring Harbor Laboratory Press p.87, 1991). Evidence suggests that these five proteins are different fromany of these known proteins inducing apoptosis as described below:

[0188] (1) The activities are different. In our assays, TNF and TGFinduced apoptosis in liver cancer cells without effect in prostatecancer (LNCAP) cells even if a very high dose (100 ng/ml) is used.

[0189] Whereas Apogen P-1's and Apogen P-2 induced apoptosis in prostatecancer rather than in cancer liver cells.

[0190] (2) TRAIL and Fas are membrane bound proteins, (Wiley, S. R. etal. Immunity 3:673, 1995; Krammer, et al., “Apoptosis in the APO-1System”, Apoptosis: The molecular Basis of Cell Death, Cold SpringHarbor Laboratory Press, p. 87, 1991) whereas the Apogen P-1a, ApogenP-1b, Apogen P-1c, Apogen P-2 and Apogen L are all soluble (non-membranebound) proteins.

[0191] (3) The molecular weights of TNF, TGF and Fas ligand TRAIL arearound 17-40 Kd (TNF=17 KD, TGF=24 KD, TRAIL=32 KD, Fas ligand=43 KD)(McGrath, M. H. Clinics in Plastic surgery 17:421, 1993; Wiley, S. R. etal., Immunity 3:673, 1995; Krammer, et al., “Apoptosis in the APO-1System”, Apoptosis: The molecular Basis of Cell Death, Cold SpringHarbor Laboratory Press, p. 87, 1991) whereas the molecular weights ofApogen P-1a, Apogen P-1b, Apogen P-1c, Apogen P-2 and Apogen L arebetween 55-70 Kd.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 7 <210> SEQ ID NO 1 <211>LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Bovine <222> LOCATION:300..309 <223> OTHER INFORMATION: Polypeptide fragment from treatment offetuin from bovine sera as described in the specification. <400>SEQUENCE: 1 His Thr Phe Ser Gly Val Ala Ser Val Glu 1 5 10 <210> SEQ IDNO 2 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Bovine <222>LOCATION: 311..317 <223> OTHER INFORMATION: Polypeptide fragment fromtreatment of fetuin from bovine sera as described in the specification.<400> SEQUENCE: 2 Ser Ala Ser Gly Glu Ala Phe His 1 5 <210> SEQ ID NO 3<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Human <222> LOCATION:300..309 <223> OTHER INFORMATION: Polypeptide fragment from fetuin.<400> SEQUENCE: 3 His Thr Phe Met Gly Val Val Ser Leu Gly 1 5 10 <210>SEQ ID NO 4 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Pig <222>LOCATION: 300..309 <223> OTHER INFORMATION: Polypeptide fragment fromfetuin. <400> SEQUENCE: 4 His Ser Phe Ser Gly Val Ala Ser Val Glu 1 5 10<210> SEQ ID NO 5 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Sheep<222> LOCATION: 300..309 <223> OTHER INFORMATION: Polypeptide fragmentfrom fetuin. <400> SEQUENCE: 5 His Thr Phe Ser Gly Val Ala Ser Val Glu 15 10 <210> SEQ ID NO 6 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:Rat <222> LOCATION: 300..309 <223> OTHER INFORMATION: Polypeptidefragment from fetuin. <400> SEQUENCE: 6 His Thr Phe Ser Gly Val Ala SerVal Glu 1 5 10 <210> SEQ ID NO 7 <211> LENGTH: 10 <212> TYPE: PRT <213>ORGANISM: Mouse <222> LOCATION: 300..309 <223> OTHER INFORMATION:Polypeptide fragment from fetuin. <400> SEQUENCE: 7 His Ala Phe Ser ProVal Ala Ser Val Glu 1 5 10

1-10. (Cancelled)
 11. A compound for the treatment of colon and prostatecancer selected from the group consisting of polypeptides having aminoacid sequences of H-T-F-S-G-V-A-S-V-E, H-T-F-M-G-V-V-S-L-G,H-A-F-S-P-V-A-S-V-E, H-S-F-S-G-V-A-S-V-E, and analogs thereof.
 12. Afetuin polypeptide comprising an amino acid sequence ofH-T-F-S-G-V-A-S-V-E, wherein said polypeptide causes apoptosis in colonand prostate cancer cells.
 13. A fetuin polypeptide comprising an aminoacid sequence of H-T-F-M-G-V-V-S-L-G, wherein said polypeptide causesapoptosis in colon and prostate cancer cells.
 14. A fetuin polypeptidecomprising an amino acid sequence of H-A-F-S-P-V-A-S-V-E, wherein saidpolypeptide causes apoptosis in colon and prostate cancer cells.
 15. Afetuin polypeptide comprising an amino acid sequence ofH-S-F-S-G-V-A-S-V-E, wherein said polypeptide causes apoptosis in colonand prostate cancer cells.